Commonly, contemporary methods for separation of components from blood plasma or other blood based material, including IgG, Albumin and Alpha-1-trypsion depend upon early work by Edwin J. Cohn. As found in U.S. Pat. No. 5,177,194 issued Jan. 5, 1993 to Maria E. Sarno, et al. (SARNO), “One scheme in widespread use is the well-known Cohn fractionation method, which is based on differential precipitation using cold ethanol.” Cohn et al. J. Am. Chern. Soc. 68, 459 (1946).
A U.S. Pat. No. 2,390,074 issued Dec. 4, 1945 to Edwin J. Cohn (Cohn) disclosed use of alcohol, acetone and dioxane as precipitants in such fractionation processes. Continued dependence upon alcohol as a precipitant is further demonstrated in U.S. Pat. No. 6,893,639 B2 issued May 17, 2005 to Joshua Levy, et al. (Levy), wherein it is stated, “The conventional industrial methods of immune globulins purification from blood plasma are based on cold ethanol fractionation which co-precipitate groups of proteins based on their isoelectric points at given alcohol concentration at sub-zero temperatures.”
Cohn's work was stimulated by the need of the military for a stable solution for use as a plasma volume expander during World War II to replace lyophilized plasma. Consequently, the Cohn method focused on optimizing the process for separating the albumin fraction which provides the osmolality necessary for plasma volume expansion.
Even so, the use of alcohol precipitants is not without difficulties, as illustrated by Cohn, “Some protein precipitants, such as alcohol, have a tendency to denature many proteins with which they come in contact, the danger of denaturation increasing with concentration of the alcohol and increase in temperature. For many proteins, it has been found advisable to exercise considerable care in mixing the precipitant with the plasma or other protein solution in order to avoid denaturation of the protein.” It is known in the art that alcohol is a denaturant of gamma globulins, but alcohol it is greater a denaturant of alpha-1-trypsin, and for this reason, it is considered prudent to provide an alcohol-free method fractionating alpha-1-trypsin.
Further considerations of combining ethanol and water may be warranted relative to denaturation of proteins. For example, if one adds 500 ml of ethanol (100%) to 500 ml of water, one does not obtain 1000 ml of 50% ethanol. Rather, the final volume is approximately 956 ml. It is surmised that the reduction in volume is due to a tight binding between the ethanol and water molecules. Such binding may be a cause of changes in protein configuration resulting in some permanent denaturation of protein molecules which remains after ethanol is removed and water is returned.
In the 1970's, chromatography was found to be useful in the separation and purification of plasma proteins. Chromatography separates plasma proteins by specifically targeting unique characteristics of each, including molecular size (gel filtration), charge (ion exchange chromatography), and known interactions with specific molecules (affinity chromatography).
The use of various chromatographic methods on an industrial scale has been adopted for the isolation of small-weight, high-value proteins, such as Factor VIII, from plasma, and for the final purification of gamma globulin after separation from the plasma by Cohn, or modified Cohn methodologies. However, chromatographic separation of the large-weight, lower-value fractions such as albumin and gamma globulin, on an industrial scale has not been found to be practical.
Two U.S. patent applications, filed by Edward Shanbrom, having Application Numbers 20030022149 (Shanbrom '149) and 20030129167 (Shanbrom '167) filed Jan. 30, 2003 and Jul. 10, 2003, respectively, teach of use of carboxylic salts (e.g., trisodium citrate) as an agent for enhancing formation of a cryoprecipitate from plasma. The method(s) of Shanbrom generally involve trisodium citrate and other citrate salts as agents for enhancing production of blood clotting factors from cryoprecipitate.
Shanbrom '149 teaches in paragraph 0009 that “It is an object of the present invention to provide enhanced yields of cryoprecipitate.” Shanbrom also teaches, in paragraph 0011, that carboxylic acids are effective agents for enhancing the production of blood clotting factors from the cryoprecipitate. Shanbrom '149 notes that the addition of citrate to plasma, especially at concentrations between two and ten percent, by weight, does not appreciably denature labile proteins. Moreover, it is noted in Shanbrom '149 that citrate potentiates or enhances the killing of microorganisms by heat treatment.
Shanbrom '167 notes in paragraph 0015 that, “Not only does added citrate increase the amount of cryoprecipitate, it simplifies the process by decreasing the requirement for freezing . . . ” plasma in order to harvest cryoprecipitate. Shanbrom clearly teaches use of production of a cryoprecipitate for the purpose of fractionating products from the cryoprecipitate through the use of trisodium citrate in concentrations of two to ten percent.
In the 1950's, it was discovered that a “cryoprecipitate” derived from blood-based material, contained various factors was useful in treating clotting disorders such as hemophilia. Such a cryoprecipitate, as the name implies, was obtained by freezing blood plasma followed by controlled thawing at zero to four degrees Centigrade to form a liquid suspension of the precipitate. A cryo-poor supernatant derived from the cryo-precipitation process was then available for fractionation using methods according to Cohn to produce albumin and gamma globulin. Subsequent developments led to fractionation of cryoprecipitate into pure concentrates of Factor VIII, von Willebrand Factor, and other clotting factors.
While Shanbrom '149 and '167 deal directly with extracting labile coagulation products from a cryoprecipitate formed through use of citrate compounds, particularly trisodium citrate, and with killing microorganisms in the cryoprecipitate using the citrate compounds, the instant invention deals directly with extracting non-labile products (e.g., albumin, gamma globulin and alpha-1-antitrypsin) from cryo-poor plasma through use of salt compounds. Shanbrom fails to teach a cryo-poor plasma in any way.
These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints, and open-ended ranges should be interpreted to include commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.